Device and method for multiple analyte detection

ABSTRACT

The invention is directed to a method and device for simultaneously testing a sample for the presence, absence, and/or amounts of one or more a plurality of selected analytes. The invention includes, in one aspect, a device for detecting or quantitating a plurality of different analytes in a liquid sample. The device includes a substrate which defines a sample-distribution network having (i) a sample inlet, (ii) one or more detection chambers, and (iii) channel means providing a dead-end fluid connection between each of the chambers and the inlet. Each chamber may include an analyte-specific reagent effective to react with a selected analyte that may be present in the sample, and detection means for detecting the signal. Also disclosed are methods utilizing the device.

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation of Ser. No. 09/627,580 filedJul. 28, 2000, which is a continuation of Ser. No. 09/012,045 filed Jan.22, 1998, now U.S. Pat. No. 6,124,138, which is a division of Ser. No.08/831,983 filed Apr. 2, 1997, now U.S. Pat. No. 6,126,899, which claimsthe benefit of priority of U.S. Provisional Application Ser. No.60/014,712 filed Apr. 3, 1996, all of which are incorporated herein byreference.

REFERENCES

[0002] Ausubel, F. M., et al., Current Protocols in Molecular Biology,John Wiley & Sons, Inc., Media, Pa.

[0003] Bergot, J. B., et al., PCT Pub. No. WO 91/05060 (1991).

[0004] Blake, et al., Biochemistry 24:6132 (1985a).

[0005] Blake, et al., Biochemistry 24:6139 (1985b).

[0006] Buchardt, O., et al., PCT Pub. No. WO 92/20703 (1992).

[0007] Froehler, et al., Nucl. Acids Res. 16:4831 (1988).

[0008] Fung, S., et al., EPO Pub. No. 233,053 A2 (1987).

[0009] Higuchi, R., et al., Bio/Technology 10:413 (1992).

[0010] Higuchi, R., et al., Bio/Technology 11:1026 (1993).

[0011] Ishiguro, T., et al., Anal. Biochem. 229:207 (1995).

[0012] Kornberg, A., et al., DNA Replication, pp 46-47, W. H. Freemanand Co., New York (1992).

[0013] Landegren, U., et al., Science 241:1077 (1988).

[0014] Lee, L. G., et al. Nucl. Acids Res. 21:3761 (1993).

[0015] Livak, K. J.., et al. PCR Methods and Applications 4:357 (1995).

[0016] Menchen, S. M., et al., U.S. Pat. No. 5,188,934 (1993).

[0017] Menchen, S. M., et al., PCT Pub. No. WO 94/05688 (1994).

[0018] Miller, P. S., et al, Biochemistry 18:5134 (1979).

[0019] Miller, P. S., et al., J. Biol. Chem. 255:6959 (1980).

[0020] Miller, P. S., et al., Bioconj. Chem. 1:187 (1990).

[0021] Mullis, K., U.S. Pat. No. 4,683,202 (1987).

[0022] Murakami, et al., Biochemistry 24:4041 (1985).

[0023] Saiki, R. K., et al., Science 230:1350 (1985).

[0024] Sambrook, J., et al., Molecular Cloning, 2nd Ed., Cold SpringHarbor Laboratory Press, NY (1989).

[0025] Segev, D., PCT Pub. No. WO 90/01069 (1990).

[0026] Segev, D., “Amplification of Nucleic Acid Sequences by the RepairChain Reaction” in Nonradioactive Labeling and detection ofBiomolecules, C. Kessler (Ed.), Springer Laboratory, Germany (1992).

[0027] Stirchak, E. P., et al., Org. Chem. 52:4202 (1987).

[0028] Sze, S. M., ed., VLSI Technology, 2nd Ed., McGraw-Hill Pub., NewYork, N.Y. (1988).

[0029] Ullman, E. F., U.S. Pat. No. 4,161,515 (1979).

[0030] Ullman, E. F., et al., U.S.. Pat. No. 4,261,968 (1981).

[0031] Whiteley, N. M., et al., U.S. Pat. No. 4,883,750 (1989).

[0032] Winn-Deen, E., et al., Clin. Chem. 37: 1522 (1991).

[0033] Yoshida, et al., U.S. Pat. No. 4,233,401 (1980).

BACKGROUND OF THE INVENTION

[0034] Biochemical testing is becoming an increasingly important toolfor detecting and monitoring diseases. While tests have long been knownfor obtaining basic medical information such as blood type andtransplant compatibility, for example, advances in understanding thebiochemistry underlying many diseases have vastly expanded the number oftests which can be performed. Thus, many tests have become available forvarious analytical purposes, such as detecting pathogens, diagnosing andmonitoring disease, detecting and monitoring changes in health, andmonitoring drug therapy.

[0035] An important obstacle which has limited exploitation of manybiochemical tests has been cost. Simultaneous testing of multiplesamples for a single analyte has provided some savings. However,simultaneous assays for a large number of analytes within a singlesample have been less practical because of the need for extended samplemanipulation, multiple test devices, multiple analytical instruments,and other drawbacks.

[0036] Ideally, a method for analyzing an individual sample using asingle test device should provide diagnostic information for a largenumber of potential analytes while requiring a small amount of sample.The device should be small in size while providing high-sensitivitydetection for the analytes of interest. The method should also requireminimal sample manipulation. Preferably, the device will includepre-dispensed reagents for specific detection of the analytes.

SUMMARY OF THE INVENTION

[0037] The present invention is directed generally to a method anddevice for simultaneously testing a sample for the presence, absenceand/or amount of one or more selected analytes.

[0038] The invention includes, in one aspect, a device for detecting orquantitating one or more of a plurality of different analytes in aliquid sample. The device includes a substrate which defines asample-distribution network having (i) a sample inlet, (ii) one or moredetection chambers, and (iii) channel means providing a dead-end fluidconnection between each of the chambers and the inlet. Preferably, eachchamber includes an analyte-specific reagent effective to react with aselected analyte that may be present in the sample, and detection meansfor detecting the signal.

[0039] In one embodiment, the detection means for each chamber includesan optically transparent window through which the signal can be detectedoptically. In another embodiment, the detection means includes anon-optical sensor for detecting the signal.

[0040] The channel means of the device may be configured in numerousways. For example, in one embodiment, the channel means includes asingle channel to which the detection chambers are connected by dead-endfluid connections. In a second embodiment, the channel means includes atleast two different channels, each connected to a different group ofdetection chambers. In yet another embodiment, the channel meansincludes an individual channel for each detection chamber.

[0041] The device may include a vacuum port for placing the detectionchambers under vacuum prior to the addition of sample. In oneembodiment, the vacuum port is connected to the channel means at a sitebetween, and in fluid communication with, the sample inlet and thedetection chambers. In another embodiment, the vacuum port is connectedto the channel means at a site downstream of the detection chambers. Inthis configuration, the vacuum port is additionally useful for removingliquid from the channel means after the detection chambers have beenfilled, to help isolate the detection chambers from one another andfurther reduce cross-contamination.

[0042] The vacuum port may be incorporated in a multi-port valve (e.g.,a 3-way valve) that permits the network and associated detectionchambers to be exposed alternately to a vacuum source, the sample inlet,and a vent or selected gas source.

[0043] Alternatively, the device of the invention is prepared and sealedunder vacuum when manufactured, so that a vacuum port is unnecessary.

[0044] According to an important feature of the invention, the device iscapable of maintaining a vacuum within the sample-distribution network(low internal gas pressure, relative to the external, ambient pressureoutside the device) for a time sufficient to allow a sample to be drawninto the network and distributed to the detection chambers by vacuumaction. For this purpose, the sample-distribution network may include avacuum reservoir in fluid communication with, and downstream of, thedetection chambers, for preventing the build-up of back-pressure in thenetwork while the detection chambers are successively filled.

[0045] In one embodiment, the vacuum reservoir includes anon-flowthrough cavity connected downstream of the last-filled detectionchamber, for accumulating residual gas displaced from the inlet andchannel means. In another embodiment, the reservoir comprises theterminal end of the channel means connected to a vacuum source, allowingresidual gas displaced by the sample to be removed continuously untilsample loading is complete.

[0046] The analyte-specific reagents in the detection chambers may beadapted to detect a wide variety of analyte classes, includingpolynucleotides, polypeptides, polysaccharides, and small moleculeanalytes, for example. In one embodiment, the analytes areselected-sequence polynucleotides, and the analyte-specific reagentsinclude sequence-selective reagents for detecting the polynucleotides.The polynucleotide analytes are detected by any suitable method, such aspolymerase chain reaction, ligase chain reaction, oligonucleotideligation assay, or hybridization assay.

[0047] In one particular embodiment, for polynucleotide detection, theanalyte-specific reagents include an oligonucleotide primer pairsuitable for amplifying, by polymerase chain reaction, a targetpolynucleotide region in the selected analyte which is flanked bysequences complementary to the primer pair. The presence of targetpolynucleotide, as indicated by successful amplification, is detected byany suitable means.

[0048] In another embodiment, the analyte-specific reagents in eachdetection chamber include an antibody specific for a selectedanalyte-antigen. In a related embodiment, when the analyte is anantibody, the analyte-specific detection reagents include an antiben forreacting with a selected analyte antibody which may be present in thesample.

[0049] In yet another embodiment, the device includes means forregulating the temperatures of the detection chambers, preferablyproviding temperature control between 0° C. and 100° C., for promotingthe reaction of the sample with the detection reagents. In one preferredembodiment, the temperature regulating means includes a conductiveheating element for each detection chamber, for rapidly heating thecontents of the chamber to a selected temperature. The temperaturecontrol means is preferably adapted to regulate the temperatures of thedetection chambers, for heating and cooling the chambers in accordancewith a selected assay protocol.

[0050] The device may be manufactured and packaged so that thesample-distribution network (e.g., sample inlet, detection chambers, andchannel means) is provided under vacuum, ready for immediate use by theuser. Alternatively, the sample-distribution network is provided underatmospheric pressure, so that the evacuation step is carried out by theend-user prior to sample loading.

[0051] The invention also includes a substrate containing a plurality ofsample-distribution networks as described above, for testing a singlesample or a plurality of samples for selected analytes.

[0052] In another aspect, the invention includes a method of making adevice such as described above.

[0053] In another aspect, the invention includes a method for detectingor quantitating a plurality of analytes in a liquid sample. In themethod, there is provided a device of the type described above, whereinthe interior of the network is placed under vacuum. A liquid sample isthen applied to the inlet, and the sample is allowed to be drawn intothe sample-distribution network by vacuum action, delivering sample tothe detection chambers. The delivered sample is allowed to react withthe analyte-specific reagent in each detection chamber under conditionseffective to produce a detectable signal when the selected analyte ispresent in the sample. The reaction chambers are inspected or analyzedto determine the presence and/or amount of the selected analytes in thesample.

[0054] The device of the invention may also be provided as part of a kitwhich additionally includes selected reagents, sample preparationmaterials if appropriate, and instructions for using the device.

[0055] These and other objects and features of the invention will bemore apparent from the following detailed description when read in lightwith the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

[0056]FIGS. 1A and 1B show a plan view (1A) and perspective view (1B) ofan exemplary assay device in accordance with the invention;

[0057] FIGS. 2A-2C illustrate several exemplary sample distributionnetwork configurations in accordance with the invention;

[0058] FIGS. 3A-3C illustrates a time sequence for the filling of thedetection chambers of a sample-distribution network with fluid sample;

[0059]FIG. 4 illustrates a sample-distribution network containing threesample delivery channels for delivering sample to three different setsof detection chambers;

[0060]FIG. 5 illustrates a sample-distribution network having a separatedelivery channel for each detection chamber;

[0061] FIGS. 6A-6C illustrate selected features of anothersample-distribution network in accordance with the invention; the deviceis shown in plan view (6A), perspective view (6B), with a portion of thesample distribution network of the device shown in FIG. 6C;

[0062]FIG. 7 shows an exploded view of a portion of a device inaccordance with the invention;

[0063]FIG. 8 shows an exploded view of a portion of another device inaccordance with the invention; and

[0064]FIG. 9 shows a perspective view of another device in accordancewith the invention.

DETAILED DESCRIPTION OF THE INVENTION

[0065] I. Definitions

[0066] The following terms and phrases as used herein are intended tohave the meanings below.

[0067] “Dead-end fluid connection between a detection chamber and asample inlet” refers to a fluid connection which provides the sole fluidaccess to a detection chamber, such that fluid cannot enter or exit thedetection chamber by any other way than through the dead-end fluidconnection.

[0068] In particular, “dead-end fluid connection” refers to a channelwhose cross-section is sufficiently narrow to preclude bi-directionalfluid flow through the channel. That is, liquid cannot flow through thechannel in one direction while air or another liquid is flowing throughthe channel in the opposite direction.

[0069] As used herein, “microdevice” means a device in accordance withthe invention

[0070] II. Assay Device

[0071] In one aspect, the present invention provides a device which isuseful for testing one or more fluid samples for the presence, absence,and/or amount of one or more selected analytes. The device includes asubstrate which defines a sample-distribution network having (i) asample inlet, (ii) one or more detection chambers (preferably aplurality of detection chambers), and (iii) channel means providing adead-end fluid connection between each of the chambers and the inlet.Each chamber includes an analyte-specific reagent effective to reactwith a selected analyte that may be present in such sample.

[0072] In one embodiment, the substrate also provides, for each chamber,an optically transparent window through which analyte-specific reactionproducts can be detected. In another embodiment, the detection means foreach chamber comprises a non-optical sensor for signal detection.

[0073] The present invention provides a number of advantages in an assayfor multiple analytes in a sample, as will be discussed below. Inparticular, the invention facilitates the transition from a macro sizesample to a micro-sized sample, wherein the device of the inventionprovides one-step metering of reagents and sample in a multi-analytedetection assay.

[0074] A. Network Configurations

[0075]FIGS. 1A and 1B show a plan view and perspective view,respectively, of an exemplary assay device 30 in accordance with theinvention. Device 30 includes a substrate 32 which defines asample-distribution network 34. With reference to FIG. 1B, the devicealso includes mount 36 containing a sample inlet 38 and optionally,vacuum port means 40 which is located downstream of the detectionchambers.

[0076] Inlet 38 may be adapted to form a vacuum-tight seal with the endof a syringe, for sample loading, or with a multi-port valve to providefluid communication with the sample and one or more liquid or gaseousfluids. The inlet may further include a septum cap, if desired, formaintaining the network under vacuum and allowing introduction of sampleby canula or needle.

[0077] Vacuum port 40 may be adapted for connection to a vacuum source,such as a vacuum pump. The vacuum connection may include a valve forclosing off the sample-distribution network from the vacuum source, or amulti-port valve for connection to a vacuum source and one or moreselected gas supplies.

[0078] Substrate 30 further provides indentations or holes 42, which maybe arranged asymmetrically as illustrated in FIG. 1A, to engagecorresponding pins or protrusions in a device-holder, not shown, toimmobilize and orient the device for analysis.

[0079] As noted in the Summary of the Invention, the sample-distributionnetwork of the invention may utilize any of a number of differentchannel configurations, or channel means, for delivering sample to theindividual detection chambers. With reference to FIG. 2A, distributionnetwork 34 a includes sample inlet 38 a, a plurality of detectionchambers 44 a, and channel means comprising a single channel 46 a towhich the detection chambers are each connected by dead-end fluidconnections 48 a. The detection chambers are distributed on either sideof channel 44 a, with the fluid connections branching off in pairs fromopposite sides of the channel. FIG. 2B shows a portion of an alternativenetwork 34 b having an inlet 38 b and detection chambers 44B, whereinfluid connections 48 b branch off from channel 46 b in a staggeredmanner.

[0080] The detection chambers in the device of the invention may bearranged to form a repeating 2-dimensional array which facilitatesindexing and identification of the various chambers, as well as allowingrapid measurement of an optical signal produced by each chamber uponreaction with the sample, if optical detection is used.

[0081] FIGS. 2A-2B, for example, show networks in which the detectionchambers are arranged in rows and columns along perpendicular axes,allowing the chambers to be identified by X and Y indices if desired.This type of array (a perpendicular array) also facilitates successiveinterrogations of the chambers in a chamber-by-chamber analysis mode.However, other arrangements may be used, such as a staggered or aclose-packed hexagonal array. FIG. 2C, for example, shows part of anetwork 34 c having inlet 38 c and an array of staggered detectionchambers 44 c. The detection chambers are connected to a common deliverychannel 46 c by fluid connections 48 c.

[0082] The device may also include identifying symbols adjacent thedetection chamber to facilitate identification or confirmation of theanalytes being detected.

[0083] Preferably, the detection chambers of the device are eachprovided with analyte-specific reagents which are effective to reactwith a selected analyte which may be present in the sample, as discussedfurther below. Reaction of the sample with the analyte-specific reagentsresults in production of a detectable signal which indicates that theselected analyte is present.

[0084] According to an important feature of the invention, the sample isdelivered to the detection chambers by vacuum action. Prior to loadingwith sample, the interior of the sample-distribution network is placedunder vacuum so that the residual gas pressure in the network issubstantially below atmospheric pressure (i.e., substantially less than760 mm Hg). One advantage of this feature of the invention is that apump for pushing fluid through the network is not required. Instead, thedevice exploits ambient atmospheric pressure to push the sample throughthe sample inlet and into the sample-distribution network. This allowsthe sample to be delivered quickly and efficiently to the detectionchambers.

[0085] FIGS. 3A-3A illustrate the filling process for asample-distribution network 34 in accordance with FIG. 2A. The networkincludes sample inlet 38, detection chambers 44, and sample deliverychannel 46 which is connected to the various detection chambers bydead-end fluid connections 48. The network further includes a vacuumreservoir 40 at the terminus of the delivery channel. A plurality of thedetection chambers 44 contain dried detection reagents for detecting adifferent selected analyte in each chamber, with one or more chambersoptionally being reserved as controls.

[0086]FIG. 3A shows the device before sample loading is initiated. Thenetwork is evacuated to establish an internal pressure within thenetwork that is substantially below atmospheric pressure (e.g., 1 to 40mm Hg). The interior of the network should also be substantiallyliquid-free to minimize vapor pressure problems. FIG. 3B shows thenetwork after sample fluid 50 has entered the network through inlet 38(FIG. 3B). As the sample moves through channel 46, the samplesequentially fills each of the detection chambers (FIG. 3B) until all ofthe chambers have been filled (FIG. 3C). With continued reference toFIG. 3C, once the detection chambers have all been filled, sample fluidmay continue to flow through channel 46 into vacuum reservoir 40 untilthe reservoir becomes full or the flow is otherwise terminated (e.g., byclosing a valve associated with the vacuum reservoir).

[0087] According to one advantage of the invention, continued sampleflow through the channel means does not substantially disturb thecontents of the detection chambers that have already been filled,because further flow into or out of each filled detection chamber isrestricted by the dead-end fluid connections, such as connections 48.Cross-contamination between different detection chambers is thereforereduced, so that erroneous signals due to cross-contamination can beavoided. A further advantage of the invention is that the sample can bemixed with the analyte-specific detection reagents and detected all inthe same chamber, without requiring movement of the sample from eachchamber to another site. Moreover, since the sample and detectionreagents can remain in the chamber for signal detection, the detectionreagents need not be immobilized on or adhered to the inner surfaces ofthe detection chambers.

[0088] The components of the sample-distribution network are designed toensure that an adequate volume of sample will be delivered to thedetection chambers to allow accurate analyte detection and/orquantitation. In general, the percent-volume of a detection chamber thatmust be occupied by the sample will vary according to the requirementsof the reagents and the detection system used. Typically, thevolume-percent will be greater than 75%, preferably greater than 90%,and more preferably greater than 95%. In assay formats in which thedetection chambers are heated, particularly to temperatures of betweenabout 60° C. and about 95° C., the volume-percent filling of thechambers is preferably greater than 95%, and more preferably is at least99%.

[0089] The degree to which the detection chambers are filled with samplewill generally depend upon (1) the initial ratio of the external(atmospheric) pressure to the initial pressure within the network, (2)the individual and total volumes defined by the detection chambers, (3)the volume defined by the channel means, and (4) the nature of thenetwork downstream of the last detection chamber.

[0090] For example, in the case of a detection chamber which is nearestthe sample inlet, and which will be filled first, the percentageoccupancy (volume-percent) of sample fluid in the chamber after sampleloading (V_(5,%)) will be related to the external atmospheric pressure(P_(ext)) and the initial internal pressure within the network beforesample loading (P_(int)) by the expression:

V _(5,%)˜(P _(ext))/(P _(ext) +P _(int))

[0091] Thus, if the initial pressure within the network (P_(int)) is 10mm Hg, and the external pressure (P_(ext)) is 760 mm Hg, about 99% ofthe first detection chamber will be filled with sample fluid(V_(5,%)˜99%), with the remaining volume (˜1.3%) being filled byresidual gas (e.g., air) displaced by the sample. (This calculationassumes that, by the time the sample reaches the chamber, the internalnetwork pressure has not increased appreciably due to displacement ofgas upstream of the chamber.) Similarly, if P_(ext) is 760 mm Hg andP_(int) is only 40 mm Hg, the volume-percent of the chamber that becomesoccupied with sample will still be very high (about 95%).

[0092] It will be appreciated that as the sample fluid reaches and fillssuccessive detection chambers, the residual gas displaced from thechannel means will gradually accumulate in the remaining network volume,so that the internal pressure will gradually increase. The resultantincrease in back-pressure can lead to a reduction in V_(5,%) for eachsuccessive chamber, with V_(5,%) for the last-filled detection chamberbeing significantly lower than the V_(5,%) for the first-filled chamber.

[0093] To help avoid this problem, the dimensions of the channel anddead-end fluid connections are preferably selected to define a totalvolume that is substantially less than the total volume defined by thedetection chambers. Preferably, the collective volume of the channelmeans is less than 20% of the total collective volume of the detectionchambers, and more preferably less than 5%. Similarly, the volume ofeach dead-end fluid connection should be substantially less than thevolume of the associated detection chamber. Preferably, the volume ofeach dead-end connection is less than 20%, preferably less than 10%, andmore preferably less than 5% of the volume of the associated detectionchamber.

[0094] The problem of back-pressure can be further diminished byincluding a vacuum reservoir downstream of the last detection chamber tobe filled. In one embodiment, the vacuum reservoir is a non-flowthroughcavity in which gas displaced by the sample fluid can collect. Thevolume of the reservoir will vary according to the configuration andneeds of the particular device. For example, the volume of the reservoircan be selected to be equal in volume to one or more detection chambersvolumes, or alternatively, is one- to five-fold as great as the totalcollective volume of the channel means.

[0095] In another embodiment, the vacuum reservoir is connected to avacuum source, so that residual gas can be removed continuously untilsample loading into the detection chambers is complete, as discussedfurther below.

[0096]FIG. 4 shows another sample-distribution network in accordancewith the invention, wherein the channel means of the network includes atleast two different sample delivery channels, each connected to adifferent group of detection chambers. FIG. 4 shows asample-distribution network 60 having a sample inlet 62, three differentgroups of detection chambers 64 a, 64 b, and 64 c, and channel means 66which include corresponding channels 66 a, 66 b, and 66 c associatedwith the three chamber groups. The chambers are connected to channels 64a-64 c via dead-end fluid connections 68 a-68 c, which provideuni-directional flow of the sample into the detection chambers.

[0097] One advantage of using multiple delivery channels is that thetime needed to fill the detection chambers with the sample can besignificantly reduced relative to the time needed to fill the samenumber of detection chambers using a single delivery channel. Forexample, the loading time for a network in accordance with FIG. 4 willbe about one-third of that needed to fill an identical number ofdetection chambers via the single channel format illustrated in FIG. 2A,all other factors being equal. More generally, for a given number ofdetection chambers, the filling time will vary inversely with the numberof delivery channels used.

[0098] The sample-distribution network in FIG. 4 further includesseparate vacuum reservoirs 70 a-70 c which are connected to the terminiof sample delivery channels 64 a-64 c, downstream of the detectionchambers. The vacuum chambers are dimensioned to help maintain a lowinternal gas pressure during sample loading.

[0099] In another embodiment, the channel means includes an individualchannel for each detection chamber, as illustrated in FIG. 5. Network 80includes an inlet 82, detection chambers 84, and associated with eachdetection chamber, a dead-end fluid connection 86, which may also bereferred to as channel means, for delivering sample to each chamber.Each dead-end fluid connection is dimensioned to define a volume that issubstantially less than the volume of the associated detection chamber,to ensure that each detection chamber is sufficiently filled withsample. This embodiment provides rapid filling of the detection chamberswith minimal cross-contamination.

[0100] The device of the invention may also include a vacuum portcommunicating with the sample-distribution network, for applying avacuum to the network before or during sample loading. In oneembodiment, the vacuum port is connected to the channel means at a sitebetween, and in fluid communication with, the sample inlet and thedetection chambers. An illustration of this can be found in FIG. 9. Thevacuum port thus provides a convenient way to reduce the internalpressure within the network to a selected residual pressure prior tosample loading. In particular, when the sample is introduced into thenetwork using a syringe barrel connected to the sample inlet, the vacuumport can be used to remove air from the space between the syringe andthe inlet, before the sample is admitted into the network.

[0101] In another embodiment, the vacuum port is connected to thechannel means at a site downstream of the sample inlet and detectionchambers (e.g., FIG. 6A). In this configuration, the vacuum port mayadditionally be used to remove liquid from the channel means after thedetection chambers have been filled, to help isolate the detectionchambers from one another and further reduce cross-contamination. Inthis configuration, the vacuum port constitutes a part of the vacuumreservoir described above, where the reservoir includes a vacuum sourcelinked to the terminal end of a sample delivery channel. The vacuum portmay be kept open to the network during sample loading, to continuouslyremove residual gas from the network until all of the detection chambershave been filled.

[0102] The vacuum port may include a multi-port valve (e.g., 3-wayvalve) that permits the network and associated detection chambers to beexposed alternately to a vacuum source, the sample inlet, and a vent orgas source. Such a valve may be used to alternately expose the networkto vacuum and a selected gas source, to replace residual air with theselected gas. Such gas replacement in the network may be useful toremove molecular oxygen (O₂) or other atmospheric gases which mightotherwise interfere with the performance of the detection reagents.

[0103] Argon and nitrogen are inert gases which may be suitable for mostsituations. Another gas which may be used is carbon dioxide (CO₂), whichis highly soluble in water due to its ability to form carbonate andbicarbonate ions. When the sample fluid is an aqueous solution, bubblesof carbon dioxide which may form in the network during sample loadingmay be eliminated via dissolution in the sample fluid. The degree ofsample filling in the detection chambers is therefore enhanced ofcourse, carbon dioxide should not be used if it interferes with thedetection reagents.

[0104] A multi-port valve such as noted above can also be used to supplya gas which is required for detection of the selected analytes. Forexample, it may be desirable to provide molecular oxygen or ozone wherethe detection reagents involve an oxidation reaction. Other gases, suchas hydrocarbons (ethylene, methane) or nitrogenous gases, may also beintroduced as appropriate.

[0105] B. Device Fabrication

[0106] The device of the invention is designed to allow testing of asample for a large number of different analytes by optical analysis,using a device that is compact and inexpensive to prepare. Generally,the device will be no larger in cross-section than the cross-section ofa standard credit card (≦5 cm×10 cm), and will have a thickness (depth)of no greater than 2 cm. More preferably, the device occupies a volumeof no greater than about 5×5×1 cm, excluding attachments for the sampleinlet and any vacuum port. More preferably, the device has dimensions ofno greater than about 3 cm×2 cm×0.3 cm. Devices smaller than this arealso contemplated, bearing in mind that the device should provideadequate sensitivity and be easy for the end-user to handle.

[0107] The detection chambers in the device are generally designed to beas small as possible, in order to achieve high density of detectionchambers. The sizes and dimensions of the chambers will depend on anumber of considerations. When signal detection is by optical means, theoverhead cross-section of each chamber must be large enough to allowreliable measurement of the signal produced when the selected analyte ispresent in the sample. Also, the depths of the chambers can be tailoredfor the particular optical method used. For fluorescence detection, thinchambers may be desirable, to minimize quenching effects. For absorbanceor chemiluminescence detection, on the other hand, a thicker chamber maybe appropriate, to increase the detection signal.

[0108] It will be appreciated that while the figures in the attacheddrawings show chambers having square-shaped overhead cross-sections,other geometries, such as circles or ovals, may also be used. Similarly,the channels in the network may be straight or curved, as necessary,with cross-sections that are shallow, deep, square, rectangular,concave, or V-shaped, or any other appropriate configuration.

[0109] Typically, the detection chambers will be dimensioned to holdfrom 0.001 μL to 10 μL of sample per chamber, and, more preferablybetween 0.01 μL and 2 μL. Conveniently, the volume of each detectionchamber is between about 0.1 μL and 1 μL, to allow visual confirmationthat the chambers have been filled. For example, a chamber having avolume of 0.2 μL may have dimensions of 1 mm×1 mm×0.2 mm, where the lastdimension is the chamber's depth.

[0110] The sample delivery channels are dimensioned to facilitate rapiddelivery of sample to the detection chambers, while occupying as littlevolume as possible. Typical cross-sectional dimensions for the channelswill range from 5 μm to about 250 μm for both the width and depth.Ideally, the path lengths between chambers will be as short as possibleto minimize the total channel volume. For this purpose (to minimizevolume), the network is preferably substantially planar, i.e., thechannel means and detection chambers in the device intersect a commonplane.

[0111] The substrate that defines the sample-distribution network of theinvention may be formed from any solid material that is suitable forconducting analyte detection. Materials which may be used will includevarious plastic polymers and copolymers, such as polypropylenes,polystyrenes, polyimides, and polycarbonates. Inorganic materials suchas glass and silicon are also useful. Silicon is especially advantageousin view of its high thermal conductivity, which facilitates rapidheating and cooling of the device if necessary. The substrate may beformed from a single material or from a plurality of materials.

[0112] The sample-distribution network is formed by any suitable methodknown in the art. For plastic materials, injection molding willgenerally be suitable to form detection chambers and connecting channelshaving a desired pattern. For silicon, standard etching techniques fromthe semiconductor industry may be used, as described in Sze (1988), forexample.

[0113] Typically, the device substrate is prepared from two or morelaminated layers, as will be discussed below with reference to FIGS.6A-6C to 8. For optical detection, the device will include one or morelayers which provide an optically transparent window for each detectionchamber, through which the analyte-specific signal is detected. For thispurpose, silica-based glasses, quartz, polycarbonate, or an opticallytransparent plastic layer may be used, for example. Selection of theparticular window material depends in part on the optical properties ofthe material. For example, in a fluorescence-based assay, the materialshould have low fluorescence emission at the wavelength(s) beingmeasured. The window material should also exhibit minimal lightabsorption for the signal wavelengths of interest.

[0114] Other layers in the device may be formed using the same ordifferent materials. Preferably, the layer or layers defining thedetection chambers are formed predominantly from a material that hashigh heat conductivity, such as silicon or a heat-conducting metal. Thesilicon surfaces which contact the sample are preferably coated with anoxidation layer or other suitable coating, to render the surface moreinert. Similarly, where a heat-conducting metal is used in thesubstrate, the metal can be coated with an inert material, such as aplastic polymer, to prevent corrosion of the metal and to separate themetal surface from contact with the sample. The suitability of aparticular surface should be verified for the selected assay.

[0115] For optical detection, the opacity or transparency of thesubstrate material defining the detection chambers will generally havean effect on the permissible detector geometries used for signaldetection. For the following discussion, references to the “upper wall”of a detection chamber refer to the chamber surface or wall throughwhich the optical signal is detected, and references to the “lower wall”of a chamber refers to the chamber surface or wall that is opposite theupper wall.

[0116] When the substrate material defining the lower wall and sides ofthe detection chambers is optically opaque, and detection is byabsorption or fluorescence, the detection chambers will usually beilluminated and optically scanned through the same surface (i.e., thetop surfaces of the chambers which are optically transparent). Thus, forfluorescence detection, the opaque substrate material preferablyexhibits low reflectance properties so that reflection of theilluminating light back toward the detector is minimized. Conversely, ahigh reflectance will be desirable for detection based on lightabsorption.

[0117] When the substrate material defining the upper surface and sidesof the detection chambers is optically clear, and detection involvesfluorescence measurement, the chambers can be illuminated withexcitation light through the sides of the chambers (in the plane definedcollectively by the detection chambers in the device), or moretypically, diagonally from above (e.g., at a 45 degree angle), andemitted light is collected from above the chambers (i.e., through theupper walls, in a direction perpendicular to the plane defined by thedetection chambers). Preferably, the substrate material exhibits lowdispersion of the illuminating light in order to limit Rayleighscattering.

[0118] When the entirety of the substrate material is optically clear,or at least the upper and lower walls of the chambers are opticallyclear, the chambers can be illuminated through either wall (upper orlower), and the emitted or transmitted light is measured through eitherwall as appropriate. Illumination of the chambers from other directionswill also be possible as already discussed above.

[0119] With chemiluminescence detection, where light of a distinctivewavelength is typically generated without illumination of the sample byan outside light source, the absorptive and reflective properties of thesubstrate will be less important, provided that the substrate providesat least one optically transparent window for detecting the signal.

[0120] FIGS. 6A-6C illustrate a specific embodiment of a device inaccordance with the invention. With reference to FIGS. 6A and 6B, device100 includes a sample inlet 102, sample-distribution network 104, andvacuum port 106 which is connected to the terminus of network 104.Network 104 includes a perpendicular array of detection chambers 108 (7rows×8 columns) linked to sample delivery channel 110 via dead-end fluidconnections 112. The device further includes vertical panel 114 adjacentsample inlet 102, for attaching an identifying label to the device andas an attachment allowing the user to hold the device.

[0121] As can be seen from FIG. 6B, the detection chambers are packedclosely together to increase the number of analytes which can be testedin the device. Fluid connections 112 are provided in an L-shapedconfiguration (FIG. 6C) to impede fluid flow out of the chambers aftersample loading, and to help isolate the contents of the chambers fromeach other. Although the horizontal rows of detection chambers in FIGS.6A and 6B are shown as being separated from each other by variablevertical spacing (to enhance the clarity of the figures), it will beappreciated that the chambers can be separated by equal distances inboth the vertical and horizontal directions, to facilitate analysis ofthe chambers.

[0122]FIGS. 7 and 8 illustrate two exemplary approaches for forming atesting device in accordance with FIGS. 6A-6B. FIG. 7 shows twosubstrate layers 140 and 142 which can be brought together to formsample-distribution network 104 (FIG. 6A) The network is definedprimarily by substrate layer 140, which contains indentations defining asample inlet 102 (not shown), a plurality of detection chambers 108,sample delivery channel 110, and dead-end fluid connections 112. Contactof substrate layer 142 with the opposing face of layer 140 completes theformation of network 104.

[0123]FIG. 8 shows substrate layers 150 and 152 for forming a network byanother approach. Substrate layer 150 contains indentations defining aplurality of detection chambers 108. Substrate layer 152, on the otherhand, contains indentations defining sample delivery channel 110 anddead-end fluid connections 112. Network 104 can then be formed bycontacting the opposing faces of the two substrate layers as in FIG. 7.

[0124] Since the device is designed to provide a vacuum-tightenvironment within the sample-distribution network for sample loading,and also to provide detection chambers having carefully defined reactionvolumes, it is desirable to ensure that the network and associatedetection chambers do not leak. Accordingly, lamination of substratelayers to one another should be accomplished so as to ensure that allchambers and channels are well sealed.

[0125] In general, the substrate layers can be sealably bonded in anumber of ways. Conventionally, a suitable bonding substance, such as aglue or epoxy-type resin, is applied to one or both opposing surfacesthat will be bonded together. The bonding substance may be applied tothe entirety of either surface, so that the bonding substance (aftercuring) will come into contact with the detection chambers and thedistribution network. In this case, the bonding substance is selected tobe compatible with the sample and detection reagents used in the assay.Alternatively, the bonding substance may be applied around thedistribution network and detection chambers so that contact with thesample will be minimal or avoided entirely. The bonding substance mayalso be provided as part of an adhesive-backed tape or membrane which isthen brought into contact with the opposing surface. In yet anotherapproach, the sealable bonding is accomplished using an adhesive gasketlayer which is placed between the two substrate layers. In any of theseapproaches, bonding may be accomplished by any suitable method,including pressure-sealing, ultrasonic welding, and heat curing, forexample.

[0126] The device of the invention may be adapted to allow rapid heatingand cooling of the detection chambers to facilitate reaction of thesample with the analyte-detection reagents. In one embodiment, thedevice is heated or cooled using an external temperature-controller. Thetemperature-controller is adapted to heat/cool one or more surfaces ofthe device, or may be adapted to selectively heat the detection chambersthemselves.

[0127] To facilitate heating or cooling with this embodiment, thesubstrate material of the test device is preferably formed of a materialwhich has high thermal conductivity, such as copper, aluminum, orsilicon. Alternatively, a substrate layer such as layer 140 in FIG. 7may be formed from a material having moderate or low thermalconductivity, while substrate layer 142 (FIG. 7) is provided as a thinlayer, such that the temperature of the detection chambers can beconveniently controlled by heating or cooling the device through layer142, regardless of the thermal conductivity of the material of layer142. In one preferred embodiment, layer 142 is provided in the form ofan adhesive copper-backed tape.

[0128] In an alternative embodiment, means for modulating thetemperature of the detection chambers is provided in the substrate ofthe device itself. For example, with reference to FIG. 7, substratelayer 142 may include resistive traces which contact regions adjacentthe reaction chambers, whereby passage of electical current through thetraces is effective to heat or cool the chambers. This approach isparticularly suitable for silicon-based substrates, and can providesuperior temperature control.

[0129] Further illustration of the invention is provided by the deviceshown in FIG. 9. Device 160 includes a network-defining substrate layer161 and a flat substrate layer 180 for bonding with and sealing layer161.

[0130] Layer 161 includes sample inlet 162 and indentations defining (i)a sample-distribution network 164 and (ii) vacuum reservoir 166connected to the terminus of network 164. Network 164 includes a2-dimensional perpendicular array of detection chambers 168 (7×7) linkedto sample delivery channel 170 via dead-end fluid connections 172. Thedevice further includes vertical panel 174 adjacent sample inlet 162, asin FIGS. 6A-6B. Formation of network 164 is completed by contacting theentirety of the upper surface of device 160 with the opposing face of alayer 180, which is preferably provided in the form of a membrane orthin layer.

[0131] Device 160 in FIG. 9 is distinguished from device 100 in FIG. 6Ain that device 160 includes a vacuum reservoir 166, instead of a vacuumport, at the terminus of delivery channel 170. In addition, sample inlet162 in device 160 is conveniently adapted to operate in conjunction withinlet fitting 190, so that evacuation of the network and sample loadingcan be is effected from a single site with respect to the network.

[0132] Sample inlet 162 includes a hollow inlet cylinder 176 having anopen proximal end 177, which connects to network 172, and an open,distal end 178. Cylinder 176 further includes an opening 179 locatednear the terminus of the distal end.

[0133] Inlet fitting 190 includes an inlet cap structure 200 and a portstructure 210 appended thereto. Cap structure 200 defines a hollowcylinder 202 having an open, proximal end 204 and a closed, distal end206. The inner diameter of cylinder 202 is dimensioned to form avacuum-tight seal when placed over inlet 162. Port structure 210 definesa vacuum port 212 and a sample port 214. Ports 212 and 214 communicatewith cylinder 202 via openings 216 and 218, respectively, which areformed in the side of cylinder 202. Fitting 190 additionally includesguide structure 220 for receiving the adjacent edge of panel 174, toorient and guide fitting 190 when fitting 190 is fitted over and slidedalong inlet 162.

[0134] Exemplary dimensions of a device which has been prepared inaccordance with FIG. 9 are the following: detection chambers 168, 1.2mm×1.2 mm×0.75 mm; delivery channel 170, 0.25 mm×0.25 mm (width×depth);dead-end fluid connection 172, 0.25 mm×0.25 mm (width×depth); externaldimensions: 22 cm×15 cm×1 mm (dimensions of network-defining portion,excluding inlet 162 and panel 174). Preferably, the detection chambersin the microdevice of the invention have volumes less than 10 μL, lessthan 2 μL, and most preferably less than or equal to 1 μL.

[0135] Device 160 may be prepared under ordinary atmospheric-conditionsby bonding a polymeric, adhesive-backed substrate layer 180 to thecorresponding surface of layer 161, to form a sealed network. Inletfitting 190 is fitted onto cylinder 176 of inlet 162, such that openings179 and 216 are aligned with each other. Vacuum port 212 is connected toa vacuum line, and the interior of the network is evacuated for aselected time. The network may be alternately flushed with a selectedgas, such as carbon dioxide, and vacuum, as discussed above. Duringevacuation, sample port 214 is loaded with, or placed in fluidcommunication with, the fluid sample. Preferably, the sample port isfilled so that there is no air between the sample in the sample port andopening 218. Once the network has been evacuated (usually completewithin a few seconds), fitting 190 is lowered further towards layer 161until opening 179 is aligned with sample opening 218, bringing theinterior of the network in fluid communication with the sample. Thesample fills the chambers rapidly, typically in less than half a second.The detection chambers are filled to a volume-percent of greater than95%. Excess sample and residual gas collects in reservoir 166.

[0136] Once the chambers have filled with sample, fitting 190 is loweredfurther toward layer 161 in order to seal inlet 162, thereby sealing theinterior of the network from the outside atmosphere. The sample isallowed to react with the detection reagents in the detection chambers,during or after which the optical signals produced in the chambers aredetected.

[0137] C. Detection Reagents

[0138] The detection chamber(s) of the device may be pre-loaded withdetection reagents which are specific for the selected analytes ofinterest. The detection reagents are designed to produce an opticallydetectable signal via any of the optical methods noted in Section IIbelow.

[0139] It will be appreciated that although the reagents in eachdetection chamber must contain substances specific for the analyte(s) tobe detected in the particular chamber, other reagents necessary toproduce the optical signal for detection may be added to the sampleprior to loading, or may be placed at locations elsewhere in the networkfor mixing with the sample. Whether particular assay components areincluded in the detection chambers or elsewhere will depend on thenature of the particular assay, and on whether a given component isstable to drying. In general, it is preferred that as many of thedetection reagents as possible are pre-loaded in the detection chambersduring manufacture of the device, in order to enhance assay uniformityand minimize the assay steps conducted by the end-user.

[0140] The analyte to be detected may be any substance whose presence,absence, or amount is desireable to be determined. The detection meanscan include any reagent or combination of reagents suitable to detect ormeasure the analyte(s) of interest. It will be appreciated that morethan one analyte can be tested for in a single detection chamber, ifdesired.

[0141] In one embodiment, the analytes are selected-sequencepolynucleotides, such as DNA or RNA, and the analyte-specific reagentsinclude sequence-selective reagents for detecting the polynucleotides.The sequence-selective reagents include at least one binding polymerwhich is effective to selectively bind to a target polynucleotide havinga defined sequence.

[0142] The binding polymer can be a conventional polynucleotide, such asDNA or RNA, or any suitable analog thereof which has the requisitesequence selectivity. For example, binding polymers which are analogs ofpolynucleotides, such as deoxynucleotides with thiophosphodiesterlinkages, and which are capable of base-specific bindingto-single-stranded or double-stranded target polynucleotides may beused. Polynucleotide analogs containing uncharged, but stereoisomericmethylphosphonate linkages between the deoxyribonucleoside subunits havebeen reported (Miller, 1979, 1980, 1990, Murakami, Blake, 1985a, 1985b).A variety of analogous uncharged phosphoramidate-linked oligonucleotideanalogs have also been reported (Froehler). Also, deoxyribonucleosideanalogs having achiral and uncharged intersubunit linkages (Stirchak)and uncharged morpholino-based polymers having achiral intersubunitlinkages have been reported (U.S. Pat. No. 5,034,506). Binding polymersknown generally as peptide nucleic acids may also be used (Buchardt,1992). The binding polymers may be designed for sequence specificbinding to a single-stranded target molecule through Watson-Crick basepairing, or sequence-specific binding to a double-stranded targetpolynucleotide through Hoogstein binding sites in the major groove ofduplex nucleic acid (Kornberg). A variety of other suitablepolynucleotide analogs are also known.

[0143] The binding polymers for detecting polynucleotides are typically10-30 nucleotides in length, with the exact length depending on therequirements of the assay, although longer or shorter lengths are alsocontemplated.

[0144] In one embodiment, the analyte-specific reagents include anoligonucleotide primer pair suitable for amplifying, by polymerase chainreaction, a target polynucleotide region of the selected analyte whichis flanked by 3′-sequences complementary to the primer pair. Inpracticing this embodiment, the primer pair is reacted with the targetpolynucleotide under hybridization conditions which favor annealing ofthe primers to complementary regions of opposite strands in the target.The reaction mixture is then thermal cycled through several, andtypically about 20-40, rounds of primer extension, denaturation, andprimer/target sequence annealing, according to well-known polymerasechain reaction (PCR) methods (Mullis, Saiki).

[0145] Typically, both primers for each primer pair are pre-loaded ineach of the respective detection chambers, along with the standardnucleotide triphosphates, or analogs thereof, for primer extension(e.g., ATP, CTP, GTP, and TTP), and any other appropriate reagents, suchas MgCl₂ or MnCl₂. A thermally stable DNA polymerase, such as Taq, Vent,or the like, may also be pre-loaded in the chambers, or may be mixedwith the sample prior to sample loading. Other reagents may be includedin the detection chambers or elsewhere as appropriate. Alternatively,the detection chambers may be loaded with one primer from each primerpair, and the other primer (e.g., a primer common to all of detectionchambers) may be provided in the sample or elsewhere.

[0146] If the target polynucleotides are single-stranded, such assingle-stranded DNA or RNA, the sample is preferably pre-treated with aDNA- or RNA-polymerase prior to sample loading, to form double-strandedpolynucleotides for subsequent amplification.

[0147] The presence and/or amount of target polynucleotide in adetection chamber, as indicated by successful amplification, is detectedby any suitable means. For example, amplified sequences may be detectedin double-stranded form by including an intercalating or cross-linkingdye, such as ethidium bromide, acridine orange, or an oxazolederivative, for example, which exhibits a fluorescence increase ordecrease upon binding to double-stranded nucleic acids (Sambrook, 1989;Ausubel; Higuchi, 1992, 1993; Ishiguro, 1995).

[0148] The level of amplification can also be measured by fluorescencedetection using a fluorescently labeled oligonucleotide, such asdisclosed in Lee et al. (1993) and Livak et al. (1995). In thisembodiment, the detection reagents include a sequence-selective primerpair as in the more general PCR method above, and in addition, asequence-selective oligonucleotide (FQ-oligo) containing afluorescer-quencher pair. The primers in the primer pair arecomplementary to 3′-regions in opposing strands of the target analytesegment which flank the region which is to be amplified. The FQ-oligo isselected to be capable of hybridizing selectively to the analyte segmentin a region downstream of one of the primers and is located within theregion to be amplified.

[0149] The fluorescer-quencher pair includes a fluorescer dye and aquencher dye which are spaced from each other on the oligonucleotide sothat the quencher dye is able to significantly quench light emitted bythe fluorescer at a selected wavelength, while the quencher andfluorescer are both bound to the oligonucleotide. The FQ-oligopreferably includes a 3′-phosphate or other blocking group to preventterminal extension of the 3′-end of the oligo.

[0150] The fluorescer and quencher dyes may be selected from any dyecombination having the proper overlap of emission (for the fluorescer)and absorptive (for the quencher) wavelengths while also permittingenzymatic cleavage of the FQ-oligo by the polymerase when the is oligois hybridized to the target. Suitable dyes, such as rhodamine andfluorscein derivatives, and methods of attaching them, are well knownand are described, for example, in Menchen et al. (1993, 1994), Bergotet al. (1991), and Fung et al. (1987).

[0151] The fluorescer and quencher dyes are spaced close enough togetherto ensure adequate quenching of the fluorescer, while also being farenough apart to ensure that the polymerase is able to cleave theFQ-oligo at a site between the fluorescer and quencher. Generally,spacing of about 5 to about 30 bases is suitable, as generally describedin Livak et al. (1995). Preferably, the fluorescer in the FQ-oligo iscovalently linked to a nucleotide base which is 5′ with respect to thequencher.

[0152] In practicing this approach, the primer pair and FQ-oligo arereacted with a target polynucleotide (double-stranded for this example)under conditions effective to allow sequence-selective hybridization tothe appropriate complementary regions in the target. The primers areeffective to initiate extension of the primers via DNA polymeraseactivity. When the polymerase encounters the FQ-probe downstream of thecorresponding primer, the polymerase cleaves the FQ-probe so that thefluorescer is no longer held in proximity to the quencher. Thefluorescence signal from the released fluorescer therefore increases,indicating that the target sequence is present.

[0153] One advantage of this embodiment is that only a small proportionof the FQ-probe need be cleaved in order for a measurable signal to beproduced. In a further embodiment, the detection reagents may includetwo or more FQ-oligos having distinguishable fluorescer dyes attached,and which are complementary for different-sequence regions which may bepresent in the amplified region, e.g., due to heterozygosity (Lee,1993).

[0154] In another embodiment, the detection reagents include first andsecond oligonucleotides effective to bind selectively to adjacent,contiguous regions of a target sequence in the selected analyte, andwhich may be ligated covalently by a ligase enzyme or by chemical means(Whiteley, 1989; Landegren, 1988) (oligonucleotide ligation assay, OLA).In this approach, the two oligonucleotides (oligos) are reacted with thetarget polynucleotide under conditions effective to ensure specifichybridization of the oligonucleotides to their target sequences. Whenthe oligonucleotides have base-paired with their target sequences, suchthat confronting end subunits in the oligos are basepaired withimmediately contiguous bases in the target, the two oligos can be joinedby ligation, e.g., by treatment with ligase. After the ligation step,the detection wells are heated to dissociate unligated probes, and thepresence of ligated, target-bound probe is detected by reaction with anintercalating dye or by other means.

[0155] The oligos for OLA may also be designed so as to bring together afluorescer-quencher pair, as discussed above, leading to a decrease in afluorescence signal when the analyte sequence is present.

[0156] In the above OLA ligation method, the concentration of a targetregion from an analyte polynucleotide can be increased, if necessary, byamplification with repeated hybridization and ligation steps. Simpleadditive amplification can be achieved using the analyte polynucleotideas a target and repeating denaturation, annealing, and ligation stepsuntil a desired concentration of the ligated product is achieved.

[0157] Alternatively, the ligated product formed by hybridization andligation can be amplified by ligase chain reaction (LCR), according topublished methods (Winn-Deen). In this approach, two sets ofsequence-specific oligos are employed for each target region of adouble-stranded nucleic acid. One probe set includes first and secondoligonucleotides designed for sequence-specific binding to adjacent,contiguous regions of a target sequence in a first strand in the target.The second pair of oligonucleotides are effective to bind (hybridize) toadjacent, contiguous regions of the target sequence on the oppositestrand in the target. With continued cycles of denaturation, reannealingand ligation in the presence of the two complementary oligo sets, thetarget sequence is amplified exponentially, allowing small amounts oftarget to be detected and/or amplified.

[0158] In a further modification, the oligos for OLA or LCR assay bindto adjacent regions in a target polynucleotide which are separated byone or more intervening bases, and ligation is effected by reaction with(i) a DNA polymerase, to fill in the intervening single stranded regionwith complementary nucleotides, and (ii) a ligase enzyme to covalentlylink the resultant bound oligonucleotides (Segev, 1992, 1994).

[0159] In another embodiment, the target sequences can be detected onthe basis of a hybridization-fluorescence assay (Lee et al., 1993). Thedetection reagents include a sequence-selective binding polymer(FQ-oligo) containing a fluorescer-quencher pair, as discussed above, inwhich the fluorescence emission of the fluorescer dye is substantiallyquenched by the quencher when the FQ-oligo is free in solution (i.e.,not hybridized to a complementary sequence). Hybridization of theFQ-oligo to a complementary sequence in the target to form adouble-stranded complex is effective to perturb (e.g., increase) thefluorescence signal of the fluorescer, indicating that the target ispresent in the sample. In another embodiment, the binding polymercontains only a fluorescer dye (but not a quencher dye) whosefluorescence signal either decreases or increases upon hybridization tothe target, to produce a detectable signal.

[0160] It will be appreciated that since the selected analytes in thesample will usually be tested for under generally uniform temperatureand pressure conditions within the device, the detection reagents in thevarious detection chambers should have substantially the same reactionkinetics. This can generally be accomplished using oligonucleotides andprimers having similar or identical melting curves, which can bedetermined by empirical or experimental methods as are known in the art.

[0161] In another embodiment, the analyte is an antigen, and theanalyte-specific reagents in each detection chamber include an antibodyspecific for a selected analyte-antigen. Detection may be byfluorescence detection, agglutination, or other homogeneous assayformat. As used herein, “antibody” is intended to refer to a monoclonalor polyclonal antibody, an Fc portion of an antibody, or any other kindof binding partner having an equivalent function.

[0162] For fluorescence detection, the antibody may be labeled with afluorescer compound such that specific binding of the antibody to theanalyte is effective to produce a detectable increase or decrease in thecompound's fluorescence, to produce a detectable signal (non-competitiveformat). In an alternative embodiment (competitive format), thedetection means includes (i) an unlabeled, analyte-specific antibody,and (ii) a fluorescer-labeled ligand which is effective to compete withthe analyte for specifically binding to the antibody. Binding of theligand to the antibody is effective to increase or decrease thefluorescence signal of the attached fluorescer. Accordingly, themeasured signal will depend on the amount of ligand that is displaced byanalyte from the sample. Exemplary fluorescence assay formats which maybe adapted for the present invention can be found in Ullman (1979, 1981)and Yoshida (1980), for example.

[0163] In a related embodiment, when the analyte is an antibody, theanalyte-specific detection reagents include an antigen for reacting witha selected analyte antibody which may be present in the sample. Thereagents may be adapted for a competitive or non-competitive typeformat, analogous to the formats discussed above. Alternatively, theanalyte-specific reagents include a mono- or polyvalent antigen havingone or more copies of an epitope which is specifically bound by theantibody-analyte, to promote an agglutination reaction which providesthe detection signal.

[0164] In yet another embodiment, the selected analytes are enzymes, andthe detection reagents include enzyme-substrate molecules which aredesigned to react with specific analyte enzymes in the sample, based onthe substrate specificities of the enzymes. Accordingly, detectionchambers in the device each contain a different substrate or substratecombination, for which the analyte enzyme(s) may be specific. Thisembodiment is useful for detecting or measuring one or more enzymeswhich may be present in the sample, or for probing the substratespecificity of a selected enzyme. Particularly, preferred detectionreagents include chromogenic substrates such as NAD/NADH, FAD/FADH, andvarious other reducing dyes, for example, useful for assayinghydrogenases, oxidases, and enzymes that generate products which can beassayed by hydrogenases and oxidases. For esterase or hydrolase (e.g.,glycosidase) detection, chromogenic moieties such as nitrophenol may beused, for example.

[0165] In another embodiment, the analytes are drug candidates, and thedetection reagents include a suitable drug target or an equivalentthereof, to test for binding of the drug candidate to the target. Itwill be appreciated that this concept can be generalized to encompassscreening for substances that interact with or bind to one or moreselected target substances. For example, the assay device can be used totest for agonists or antagonists of a selected receptor protein, such asthe acetylcholine receptor. In a further embodiment, the assay devicecan be used to screen for substrates, activators, or inhibitors of oneor more selected enzymes. The assay may also be adapted to measuredose-response curves for analytes binding to selected targets.

[0166] The sample or detection reagents may also include a carrierprotein, such as bovine serum albumin (BSA) to reduce non-specificbinding of assay components to the walls of the detection chambers.

[0167] The analyte-specific detection reagents are preferably dispensedinto the detection chambers robotically using a dispensing systemdesigned to deliver small volumes of liquid solutions (e.g., 0.1 to 1μL). The system is supplied with separate analyte-specific detectionreagents which are dispensed to pre-selected detection chambers withoutcross-contamination.

[0168] A reagent loading device that has been prepared in accordancewith the invention includes a dispensing robot (Asymtek Automove 402)coupled to a plurality of drop-on-demand ink-jet printing heads. Therobot includes an X,Y-axis work table (12 inch×12 inch) having a lateralresolution of 0.001 inch, a lateral velocity of 0-20 inch/sec, a Z-axisresolution of 0.001 inch, and a Z-axis velocity of 0-8 inch/sec. Therobot optionally includes a tip locator, offset camera, strobe dropcamera, on-axis camera, and/or gravimetric drop calibration. Theprinting heads are of a drop-on-demand, piezo-electric type, havingwetted surfaces usually selected from glass, Teflon®, and polypropylene.The minimum drop volume is 25 nL, and the maximum flow is 1 μL/min.

[0169] Reagent loading is preferably accomplished undercarefully-controlled sterile conditions using one or more dedicateddispensing robots. After application, the reagents are allowed to dry inthe chambers until most or all of the solvent has evaporated. Drying maybe accelerated by baking or reduced pressure as appropriate. Thedetection chambers are then sealed by bonding the chamber containingsubstrate layer with an appropriate cover layer, and the device is readyfor use.

[0170] III. Signal Detection and Analysis

[0171] The signal produced by reaction of the analyte-specific reagentswith the sample is measured by any suitable detection means, includingoptical and non-optical methods.

[0172] Where the signal is detected optically, detection may beaccomplished using any optical detector that is compatible with thespectroscopic properties of the is signal. The assay may involve anincrease in an optical signal or a decrease. The optical signal may bebased on any of a variety of optical principals, including fluorescence,chemiluminescence, light absorbance, circular dichroism, opticalrotation, Raman scattering, radioactivity, and light scattering.Preferably, the optical signal is based on fluorescence,chemiluminescence, or light absorbance.

[0173] In general, the optical signal to be detected will involveabsorbance or emission of light having a wavelength between about 180 nm(ultraviolet) and about 50 μm (far infrared). More typically, thewavelength is between about 200 nm (ultraviolet) and about 800 nm (nearinfrared). A variety of detection apparatus for measuring light havingsuch wavelengths are well known in the art, and will typically involvethe use of light filters, photomultipliers, diode-based detectors,and/or charge-coupled detectors (CCD), for example.

[0174] The optical signals produced in the individual detection chambersmay be measured sequentially by iteratively scanning the chambers one ata time or in small groups, or may be measured simultaneously using adetector which interrogates all of the detection chambers continuouslyor at short time intervals. Preferably, the signals are recorded withthe aid of a computer capable of displaying instantaneously (inreal-time) the signal level in each of the detection chambers, and alsostoring the time courses of the signals for later analysis.

[0175] The optical signal in each chamber may be based on detection oflight having one or more selected wavelengths with defined band-widths(e.g., 500 nm±5 nm). Alternatively, the optical signal may be based onthe shape or profile of emitted or absorbed light in a selectedwavelength range. Preferably, the optical signal will involvemeasurement of light having at least two distinctive wavelengths inorder to include an internal control. For example, a first wavelength isused to measure the analyte, and a second wavelength is used to verifythat the chamber is not empty or to verify that a selected reagent orcalibration standard is present in the detection chamber. An aberrationor absence of the signal for the second wavelength is an indication thatthe chamber may be empty, that the sample was improperly prepared, orthat the detection reagents are defective.

[0176] In studies conducted in support of the invention, a detectionassembly was prepared for fluorescence detection of targetpolynucleotides in a sample using a device in accordance with theinvention. The assembly includes a translation stage for positioning thetest device. The test device includes a 7×7 array of addressabledetection chambers containing fluorescent detection reagents. Thedetector in the assembly consists of a tungsten bulb (or quartz halogenbulb, 75 W) illumination source, a CCD camera, and appropriatefocusing/collection optics. The illumination source is positioned so asto illuminate the device diagonally from above (e.g., at an inclinationangle of 45 degrees with respect to the illuminated surface). The opticsinclude two lenses separated by an emission filter. The first lenscollimates the incoming image for the emission filter, and the secondlens which re-images the filtered beam onto the CCD. The test device isplaced at the focal point of the first lens.

[0177] The CCD is a thermoelectrically cooled, instrumentation-gradefront-illuminated CCD (Princeton Instruments TEA/CCD-512TK/1). Thedetection plate of the CCD has a 512×512 array of 27 μm square pixelswhich covers the entire overhead cross-section of the test device. Thecamera head is controlled by a controller (Princeton Instruments ST-135)which communicates with a computer (Quadra 650, Apple Computers) forcollecting and processing the signal data. The system is capable ofon-chip binning of the pixels. For detection chambers having an overheadcross-section of 1 mm×1 mm, bins having a size of 2×2 pixels aresuitable. More generally, the bin size is selected on the basis of thetotal processing time that will be required, the sizes and number ofdetection chambers, sensitivity, and signal noise.

[0178] The computer in the assembly includes signal-processing softwarefor selecting an appropriate sub-region in each detection chamber fromwhich the signal is measured. Such sub-regions are selected foruniformity of incoming light, to ensure that edge regions are excluded.The signal image of the device is recorded and stored at selectedintervals, according to the requirements of the assay. Preferably, thesignal for each detection chamber is recorded as an average signal perbin for the selected sub-region in each chamber, since the size of theselected sub-region in each chamber will usually differ from chamber tochamber.

[0179] The detector optics may further be adapted to include a filterwheel for detecting fluorescence at 2 or more wavelengths.

[0180] As discussed above, the temperature of the detection chambers maybe controlled, if appropriate, by any of a number of suitable methods.In the detection assembly that was prepared in accordance with theinvention, the heating means is external to the test device (off-chipheating), and includes a temperature controller (Marlow Industries modelSE 5020) equipped with a peltier device (ITI Ferro Tec model 6300)having a ramp rate of about +4° /sec in the range of 55° C. to 95° C.For on-chip heating, where the device includes resistive tracings (or acomparable equivalent) for heating the chambers, the assembly can bemodified to provide one or two zones of resistance heating capable ofestablishing a maximum power dissipation of 25 W over a 200 mm² area;this mode can provide a ramp rate of ±10° /sec during transition from55° C. to 95° C.

[0181] The above described structure (Section IIB) for detecting ananalyte-related signal in each chamber, including the optical windowassociated with that chamber, is also referred to herein collectively asdetection means for detecting such signal.

[0182] Another type of detection means is a biosensor device capable ofdetecting the reaction of an analyte with an analyte-specific reagent ineach chamber. Amperometric biosensors suitable for use in the inventionoperate on a variety of principles. In one, the analyte being measuredis itself capable of interacting with an analyte-specific reagent togenerate an electrochemical species, i.e., a species capable of functionas an electron donor or acceptor when in contact with an electrode. Asan example, reaction of the analyte cholesterol with the reagentcholesterol oxidase generates the electrochemical species H₂O₂ which, incontact with an electrode, produces a measurable current in a circuitcontaining the electrode.

[0183] The analyte-specific reagent may be localized on a film separatedfrom the electrode surface by a permselective layer that is selectivelypermeable to the electrochemical species (and other small components inthe sample). When sample fluid is added to the biosensor, reaction ofthe analyte with the corresponding reagent produces an electrochemicalspecies whose presence and amount are quantitated by current measurementthrough the electrode.

[0184] Alternatively, the analyte-specific reagent may be a receptorwhich is specific for the analyte. Initially, the receptor sites arefilled with an analyte-enzyme conjugate. In the presence of analyte, theconjugates are displaced from the receptor, and are then free to migrateto positions close to the electrode, for production of transientelectrochemical species (such as H₂O₂ in the presence of catalase) inthe vicinity of the electrode.

[0185] Another general type of biosensor employs a lipid bilayermembrane as a gate for electrochemical species interposed between asample-fluid chamber and an electrode. The bilayer is provided withion-channel proteins which function as ion gates that can be opened byanalyte binding to the proteins. Thus, binding of analyte to the channelproteins (which serve as the analyte-specific reagent) leads to ion flowacross the membrane and detectable signal at the electrode.

[0186] Thin-film biosensors of the type mentioned above may be formed ina microchip or small-substrate format by photolithographic methods, suchas described in U.S. Pat. Nos. 5,391,250, 5,212,050, 5,200,051, and4,975,175. As applied to the present invention, the chamber walls in thesubstrate may serve as the substrate for deposition of the requiredelectrode and film layers. In addition to these layers, suitableconductive connectors connecting the electrodes to electrical leads arealso laid down.

[0187] In a typical device, each chamber contains a biosensor for agiven analyte. When sample is introduced into the device, the multiplesample analytes are then separately measured in the chambers, with theresults being reported to a processing unit to which the device iselectrically connected.

[0188] IV. Assay Method

[0189] In another aspect, the invention includes a method for detectingor quantitating a plurality of analytes in a liquid sample. In themethod, there is provided a device of the type described above, whereinthe interior of the network is placed under vacuum. A liquid sample isthen applied to the inlet, and the sample is allowed to be drawn intothe sample-distribution network by vacuum action, delivering sample tothe detection chambers. The delivered sample is allowed to react withthe analyte-specific reagent in each detection chamber under conditionseffective to produce a detectable signal when the selected analyte ispresent in the sample. The reaction chambers are inspected or analyzedto determine the presence and/or amount of the selected analytes in thesample.

[0190] The sample tested may be from any source which can be dissolvedor extracted into a liquid that is compatible with the device, and whichmay potentially contain one or more of the analytes of interest. Forexample, the sample may be a biological fluid such as blood, serum,plasma, urine, sweat, tear fluid, semen, saliva, cerebral spinal fluid,or a purified or modified derivative thereof. The sample may also beobtained from a plant, animal tissue, cellular lysate, cell culture,microbial sample, or soil sample, for example. The sample may bepurified or pre-treated if necessary before testing, to removesubstances that might otherwise interfere with analyte detection.Typically, the sample fluid will be an aqueous solution, particularlyfor polar analytes such as polypeptides, polynucleotides, and salts, forexample. The solution may include surfactants or detergents to improveanalytes solubility. For non-polar and hydrophobic analytes, organicsolvents may be more suitable.

[0191] As discussed above, the device may be manufactured and sold in aform wherein the sample-distribution network is under vacuum, so thatthe device is ready to load by the end-user. Alternatively, evacuationof the network is conducted by the user, through a vacuum port or viathe sample inlet itself.

[0192] Prior to sample loading, any gas in the network may be replacedwith another gas, according to the requirements of the assay. In apreferred embodiment, the residual gas is replaced with carbon dioxide,so that any gas bubbles that appear in the network after sample loadingare quickly dissolved by the sample fluid, particularly if the sample isan aqueous solution.

[0193] It will be appreciated that when the device includes a vacuumport downstream of the detection chambers, the sample delivery channelsin the device may be cleared of sample after the detection chambers havebeen filled, to further isolate the detection chambers from each other.The invention also contemplates filling the delivery channels with anadditional fluid, such as a mineral oil or a viscous polymer solutioncontaining agarose or other viscous material (e.g., see Dubrow, U.S.Pat. No. 5,164,055, and Menchen et al., U.S. Pat. No. 5,290,418), tosegregate the chambers from each other, or with a reagent-containingsolution which facilitates the assay.

[0194] In a particularly advantageous embodiment of the invention, alarge-volume syringe can be used to generate a vacuum inside the sampledistribution network of the device prior to loading. By “large-volume”is meant that the is volume of the syringe is greater than the totalinternal volume of the device (i.e., of the sample distributionnetwork). Preferably, the volume of the syringe is at least 20-foldgreater than the interior volume of the device. With reference to thedevice in FIG. 9, the inlet-tip of the syringe is connected to vacuumport 212. When opening 216 is aligned with opening 179, the syringe isused to draw air from the interior of the device, thereby lowering theinternal pressure. For example, if a syringe with a volume of 50 mL isused, and the internal volume of the device is 100 μL, the pressure inthe sample distribution network can be reduced by a factor of 500 (=0.1mL/50 mL). Thus, an initial internal pressure of 760 torr can be reducedto less than 2 torr. With reference to the device in FIG. 6A, thesyringe can be connected to fitting 106 or 102 using appropriateconnections, to withdraw air from the distribution network.

[0195] Accordingly, the present invention includes a kit comprising (i)a device as described above and (ii) a syringe for drawing air from theinterior of the device. The invention also includes a method of usingthe kit to detect one or more analytes in a sample, as described above.It will be appreciated that using a syringe greatly simplifies the stepof creating a vacuum inside the device, so that the device can be usedquickly or immediately without needing a mechanical vacuum pump.

[0196] V. Utility

[0197] The present invention can be used in a wide variety ofapplications. The invention can be used for medical or veterinarypurposes, such as detecting pathogens, diagnosing or monitoring disease,genetic screening, determining antibody or antigen titers, detecting andmonitoring changes in health, and monitoring drug therapy. The inventionis also useful in a wide variety of forensic, environmental, andindustrial applications, including screening drug candidates foractivity.

[0198] More generally, the present invention provides a convenientmethod for simultaneous assay of multiple analytes in a sample. Theinvention is highly flexible in its applications, being adaptable toanalysis of a wide variety of analytes and sample materials. Byproviding pre-dispensed, analyte-specific reagents in separate detectionchambers, the invention eliminates the need for complicated andtime-consuming reagent preparation.

[0199] Practice of the invention is further simplified since thedetection chambers can be loaded via a single sample inlet. The use ofuniformly sized detection chambers renders the device self-metering, inthat a precise volume of sample is delivered to each chamber. Thus, theprecision, accuracy and reproducibility of the assay are all enhanced,since the quantities and compositions of the analyte-specific reagents,the quantity of sample in the chambers, and the reaction conditions canbe carefully controlled. Moreover, very small volumes of sample arerequired since the dimensions of the sample-distribution network in thedevice can be very small.

[0200] The device may be formed from a wide variety of materials,allowing the composition of the device to be adapted to the particularreagents and conditions in the assay. Inasmuch as the device requires nomoving parts, and can be relatively small in size (typically havingdimension on the order of millimeters to centimeters), manufacture ofthe device is simplified and costs are reduced.

[0201] The features and advantages of the invention may be furtherunderstood from the following example, which is not intended in any wayto limit the scope of the invention.

EXAMPLE

[0202] The following study was performed using a polycarbonatemicrodevice substantially as shown in FIG. 9, to demonstrate detectionof a human β-actin gene by PCR (polymerase chain reaction). The assaycomponents for PCR detection were obtained from PE Applied Biosystems(Foster City, Calif., β-actin kit, part #N808-0230). The kit componentsincluded the following stock solutions:

[0203] β-actin forward primer:

[0204] 3 μM primer in 10 mM Tris-HCl, pH 8.0 (at room temperature), 1 mMEDTA

[0205] β-actin reverse primer:

[0206] 3 μM primer in 10 mM Tris-HCl, pH 8.0 (at room temperature), 1 mMEDTA

[0207] β-actin probe:

[0208] 2 μM TAMRA-labeled probe in 10 mM Tris-HCl, pH 8.0 (at roomtemperature), 1 mM EDTA

[0209] DNA sample:

[0210] 370 μg/mL human genomic DNA in 10 mM Tris-HCl, pH 8.0 (at roomtemperature), 1 mM EDTA (from Coriell Cell Repositories, Camden, N.J.)

[0211] dNTPs:

[0212] 20 mM dNTP (1 tube each for dUTP, DATP, dCTP and dGTP) inautoclaved deionized ultrafiltered water, titrated to pH 7.0 with NaOH

[0213] DNA polymerase:

[0214] “AMPLITAQ GOLD” DNA polymerase at 5 U/μL, from PE AppliedBiosystems, part #N808-0240 (PE Applied Biosystems “AMPLITAQ GOLD”Product Brochure, 1996)

[0215] “AMPERASE” UNG:

[0216] uracil-N-glycosylase at 1 U/μL, from PE Applied Biosystems, part#N808-0096

[0217] 10× “TAQMAN” buffer A:

[0218] 500 mM KCl, 100 mM Tris-HCl, 0.1 M EDTA, 600 nM Passive Reference1 (ROX), pH 8.3 at room temperature, autoclaved

[0219] MgCl₂:

[0220] 20 mM MgCl₂ in autoclaved deionized ultrafiltered water

[0221] A description of the sequences of the forward primer, the reverseprimer, and the TAMRA-labeled probe can be found in PE AppliedBiosystems “TAQMAN” PCR Reagent Protocol (1996), which also describesthe general steps of the “TAQMAN” assay technique. The forward andreverse primers were effective to produce a 297 basepair PCR product.

[0222] A flat substrate layer 180 and a substrate layer 161 were formedfrom polycarbonate by standard injection-molding methods (substratelayer 161) or from sheet stock (layer 180). The volume of each detectionchamber was 1 μL.

[0223] Detection chambers were loaded with different amounts of forwardprimer, reverse primer, and fluorescent probe as follows. To apolypropylene tube was added 0.5 mL each of β-actin forward primersolution, reverse primer solution, and fluorescent probe, to give afinal primer/probe stock solution volume of 1.5 mL. This solution wasthen loaded into alternating detection chambers in substrate layer 161using a robotically controlled microsyringe. Specifically, alternatechambers were loaded with either a 1×, 5× or 10× amount of primer/probesolution, with 1× (14 nL primer/probe stock solution) being equivalentto a final concentration in a detection chamber of 15 nM of each primerand 10 nM of fluorescent probe (after the dried chamber is subsequentlyfilled with sample), 5× (72 nL primer/probe stock solution) beingequivalent to a final concentration of 75 nM of each primer and 50 nM offluorescent probe, and 10× (145 nL primer/probe stock solution) beingequivalent to a final concentration of 150 nM of each primer and 100 nMof fluorescent probe. The amounts of primer and probe in the loadedchambers corresponded to {fraction (1/20)}, ¼, and ½ of theconcentrations used under standard reaction conditions, for the 1×, 5×,and 10× chambers, respectively. The loaded chambers produced a“checkerboard” pattern in substrate layer 161 where each loaded chamberwas separated by an intervening empty chamber.

[0224] After the loaded chambers were allowed to air-dry to dryness atroom temperature, the loaded substrate layer (161) was joined to a flatsubstrate layer 180 by ultrasonic welding. Inlet fitting 190 was thenplaced over sample inlet 162, such that opening 179 was aligned withvacuum port opening 216. The sample distribution network 164 anddetection chambers 168 were evacuated via vacuum port 212, which wasconnected to a vacuum pump, to a final internal pressure ofapproximately 1 to 10 torr.

[0225] The PCR reaction mixture (sample), without primers and probe, wasprepared from the above stock solutions to give the following finalconcentrations in the sample:

[0226] 10 mM Tris-HCl, pH 8.3

[0227] 50 mM KCl

[0228] 3.5 mM MgCl₂

[0229] 400 μM dUTP

[0230] 200 μM each DATP, dCTP, and dGTP

[0231] 0.01 U/μL uracil-N-glycosylase

[0232] 0.25 U/μL “AMPLITAQ GOLD” DNA polymerase

[0233] 0.74 ng/μL human genomic DNA template

[0234] For loading of sample into the microdevice, a micropipette loadedwith the above sample solution was placed in sample port 214 so as tominimize the deadvolume occupied by air at the tip of the pipette. Inletfitting 190 was then pressed down further to align opening 179 withopening 218, so that the sample was drawn from port 214 into thedetection chambers by vacuum action. Filling of the chambers wascomplete in less than a second.

[0235] The microdevice was then clamped to a peltier device (20 mm×20mm) glued to an aluminum heat sink. Cycling was controlled using aMarlow temperature controller (Marlow Industries Inc., Dallas, Tex.,Model No. SE 5020). A thermistor was attached to the peltier device toprovide temperature feedback (Marlow part No. 217-2228-006). Themicrodevice was thermocycled as follows:

[0236] 1) precycle: 50° C. for 2 minutes; 95° C. for 10 minutes;

[0237] 2) 40 cycles: 95° C. for 15 seconds, 60° C. for 1 minute;

[0238] 3) hold at 72° C.

[0239] Signal detection was accomplished using a fluorescence detectioninstrument consisting of a tungsten bulb for illumination and a CCDcamera and 4-color filter wheel for detection. Images of all detectionchambers (wells) were taken at the end of each thermocycle (during the60° C. step) at several wavelengths in order to monitor the increase ofthe reporter's fluorescence. Interfering fluorescence fluctuations werenormalized by dividing the emission intensity of the reporter dye by theemission intensity of the passive reference (ROX dye) for a givenchamber. The excitation wavelength was 488 nm. The reporter intensitywas measured at 518 nm, and the passive reference intensity was measuredat 602 nm.

[0240] Results. Positive fluorescent signals were detected in allchambers that had been loaded with the β-actin primers and fluorescentprobe at the 5× and 10 concentrations. Little or no signal was detectedfor chambers loaded at the 1× concentration. No detectable signal wasdetected above background for the chambers which did not contain β-actinprimer and probe, indicating that there was no cross-contaminationbetween detection chambers after 40 heat/cool cycles.

[0241] The highest final fluorescence signals were obtained in detectionchambers loaded with a 10× amount of primers and probe, with detectablesignals appearing after about 23 cycles. The 5× chambers also showeddetectable signals after cycle 23, but the final fluorescence signal wasnot as high as that for the 10× wells (due to lower probeconcentration). Thus, the β-actin gene was readily detected using primerand probe concentrations equal to ¼ and ½ of those used under ordinaryconditions. The results also show that the preloaded primers and probeswere successfully dissolved in the sample after sample loading.

[0242] Although the invention has been described by way of illustrationand example for purposes of clarity and understanding, it will beappreciated that various modifications can be made without departingfrom the invention. All references cited above are incorporated hereinby reference.

It is claimed:
 1. A method for detecting or quantitating one or more ofa plurality of polynucleotide sequences in a sample, said methodcomprising providing a device comprising a substrate defining asample-distribution network including (i) a sample inlet, (ii) two ormore detection chambers, and (iii) channel means providing a dead-endfluid connection between each of said chambers and said inlet, whereinat least two of said detection chambers each contain a different,sequence-specific polynucleotide binding polymer for detecting orquantitating different polynucleotide sequences that may be present insuch sample, to produce a detectable signal, applying a liquid sample tothe sample inlet and delivering the sample to the network by action of aforce whereby the sample is delivered to the detection chambers,allowing the delivered sample to react with at least onesequence-specific polynucleotide binding polymer in each detectionchamber under conditions effective to produce a detectable signal ineach detection chamber when a specific sequence is present in thesample, and measuring the signals produced in the reaction chambers todetect or quantitate specific target sequences in the sample.
 2. Themethod of claim 1, wherein said channel means comprises a single channelto which said detection chambers are connected by said fluidconnections.
 3. The method of claim 1, wherein said channel meanscomprises a first channel to which a first group of detection chambersare connected by such fluid connections, and a second channel to which asecond group of detection chambers are connected.
 4. The method of claim1, wherein said channel means comprises an individual channel for eachdetection chamber, for providing a dead-end fluid connection betweensaid inlet and each detection chamber.
 5. The method of claim 1, whereinsaid device further comprises a port connected to the channel means at asite in fluid communication with the sample inlet and detectionchambers.
 6. The method of claim 5, wherein said port is connected tothe channel means at a site that is downstream of said sample inlet andsaid detection chambers.
 7. The method of claim 1, wherein said devicefurther comprises a non-flowthrough reservoir in fluid communicationwith said channel means.
 8. The method of claim 1, wherein at least onebinding polymer comprises an oligonucleotide primer pair suitable foramplifying, by polymerase chain reaction, a specific polynucleotidesequence which is flanked by sequences complementary to the primer pair.9. The method of claim 8, wherein at least one binding polymer furthercomprises a fluorescer-quencher oligonucleotide capable of hybridizingto the specific polynucleotide sequence in a region downstream of one ofthe primers, for producing a detectable fluorescent signal when theselected sequence is present in the sample.
 10. The method of claim 1,wherein at least one binding polymer comprises first and secondoligonucleotides effective to bind to adjacent, contiguous regions of aspecific polynucleotide sequence.
 11. The method of claim 10, whereinthe at least one binding polymer comprises a second pair ofoligonucleotides which are effective to bind to adjacent, contiguousregions complementary to the regions bound by the first pair ofoligonucleotides, for amplification of the regions by ligase chainreaction.
 12. The method of claim 1, wherein at least one of thedetection chambers comprises an intercalating compound which produces anoptically detectable signal upon intercalating a double-strandedpolynucleotide.
 13. The method of claim 1, wherein said substratefurther comprises a temperature regulator for controlling thetemperature of each detection chamber.
 14. The method of claim 1,wherein said substrate defines at least two such sample-distributionnetworks.
 15. The method of claim 1, wherein said providing comprisesfilling said network with carbon dioxide gas.
 16. The method of claim 1,wherein at least one of the binding polymers contains a fluorescent dye.17. The method of claim 1, wherein at least one of the binding polymerscomprises a fluorescent dye moiety which produces a detectable signalupon hybridization of the binding polymer to a target polynucleotidesequence.
 18. The method of claim 1, wherein at least one bindingpolymer comprises first and second oligonucleotides effective to bind toadjacent regions of a specific polynucleotide sequence which areseparated from each other by one or more intervening bases.
 19. Themethod of claim 1, wherein the dead-end fluid connection provides solefluid access to each of said detection chambers, such that fluid cannotenter or exit the detection chamber by any other way than through thedead-end fluid connection.
 20. A method for analyzing one or morepolynucleotide sequences in a sample, said method comprising providing adevice comprising a substrate defining a (i) a sample inlet, (ii) aplurality of detection chambers, and (iii) channel structure providing afluid connection between each of said chambers and said inlet, whereintwo or more of said detection chambers contain one or moresequence-specific polynucleotide binding polymers for the analysis ofone or more polynucleotide sequences that may be present in such sample,to produce a detectable signal, applying a sample to the sample inletand delivering the sample to the detection chambers, allowing thedelivered sample to react with at least one sequence-specificpolynucleotide binding polymer in at least one of said detectionchambers under conditions effective to produce a detectable signal whena specific sequence is present in the sample, and detecting for saidsignal.
 21. A device for detecting or quantitating one or more of aplurality of different polynucleotide sequences in a liquid sample, saiddevice comprising a substrate defining a sample-distribution networkincluding (i) a sample inlet, (ii) two or more detection chambers, and(iii) channel means providing a dead-end fluid connection between eachof said chambers and said inlet, wherein at least two of said detectionchambers each contain a different, sequence-specific polynucleotidebinding polymer for detecting or quantitating different polynucleotidesequences that may be present in such sample, to produce a detectablesignal, whereby, application of such sample to said inlet and a changein pressure within said network, is effective to provide each of saidchambers with a portion of said sample therein.
 22. The device of claim21, wherein said channel means comprises a single channel to which saiddetection chambers are connected by said fluid connections.
 23. Thedevice of claim 21, wherein said channel means comprises a first channelto which a first group of detection chambers are connected by suchdead-end fluid connections, and a second channel to which a second groupof detection chambers are connected by such dead-end fluid connections.24. The device of claim 21, wherein said channel means comprises anindividual channel for each detection chamber, for providing a dead-endfluid connection between said inlet and each detection chamber.
 25. Thedevice of claim 21, which further comprises a port connected to saidchannel means at a site in fluid communication with the sample inlet anddetection chambers.
 26. The device of claim 25, wherein said port isconnected to said channel means at a site that is downstream of saiddetection chambers.
 27. The device of claim 21, which further comprisesa non-flowthrough reservoir in fluid communication with said channelmeans.
 28. The device of claim 21, wherein said detection meanscomprises an optically transparent window associated with each detectionchamber, through which such signal can be optically detected.
 29. Thedevice of claim 21, wherein at least one binding polymer includes firstand second oligonucleotide primers having sequences effective tohybridize to opposite end regions of complementary strands of a selectedpolynucleotide sequence, for amplifying the sequence by primer-initiatedpolymerase chain reaction.
 30. The device of claim 29, wherein at leastone binding polymer further comprises a fluorescer-quencheroligonucleotide capable of hybridizing to the selected polyucleotidesequence in a region downstream of one of the primers, for producing adetectable fluorescent signal when the selected sequence is present inthe sample.
 31. The device of claim 21, wherein at least one bindingpolymer comprises first and second oligonucleotides effective to bind toadjacent, contiguous regions of a selected polynucleotide sequence. 32.The device of claim 31, wherein the at least one binding polymercomprises a second pair of oligonucleotides which are effective to bindto adjacent, contiguous regions complementary to the regions bound bythe first pair of oligonucleotides, for amplification of the regions byligase chain reaction.
 33. The device of claim 21, wherein at least oneof the detection chambers additionally comprises an intercalatingcompound which produces an optically detectable signal uponintercalating a double-stranded polynucleotide.
 34. The device of claim21, wherein said substrate further comprises a temperature regulator forcontrolling the temperature of each detection chamber.
 35. The device ofclaim 21, wherein said substrate defines at least two suchsample-distribution networks.
 36. The device of claim 21, wherein theinterior of said network is under vacuum.
 37. The device of claim 21,wherein at least one of the binding polymers contains a fluorescent dye.38. The device of claim 21, wherein at least one binding polymercontains a fluorescent dye moiety which produces a detectable signalupon hybridization of the binding polymer to a target polynucleotidesequence.
 39. The device of claim 21, wherein at least one bindingpolymer comprises first and second oligonucleotides effective to bind toadjacent regions of a selected polynucleotide sequence which areseparated from each other by one or more intervening bases.